There is an urgent need for rapid, highly sensitive, specific, easy to use, and cost-effective medical diagnostic tests to diagnose individuals exposed to and/or infected with healthcare-associated pathogens that are pre-symptomatic, symptomatic, or have non-specific symptoms, and to accurately identify infectious agents or toxins in clinical samples so appropriate therapeutic intervention can be executed. As antibiotic resistance is spreading rapidly throughout the world causing growing costs in terms of human lives and healthcare expenditures, concomitant determination of increasingly complex microbial resistance patterns is of critical importance. However, as patient samples are limited and pathogen concentrations often extremely low (<10 organisms/ml), serial analysis is not practical, thus requiring highly multiplexed systems instead of individual pathogen-specific tests. Nesher Technologies, Inc.'s (NTI) long-term goal is to develop a rapid, highly multiplexed (with a capacity of >100 analytes per standard patient sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated, nucleic acid- and protein-based diagnostic system for bacterial infections, to allow quick and accurate pathogen identification in clinical samples, including acquired genetic traits such as antibiotic resistance or enhanced virulence. NTI has licensed the intellectual property for a revolutionary ultrasensitive biodetection technology with exquisite single well multiplexing potential, which was developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss). It is based on 3- color alternating laser excitation (3c-ALEX) single molecule fluorescence spectroscopy, whereby two (or three) recognition molecules are tagged with different color fluorescence dyes. Coincident confocal detection of two or three colors constitutes a positive target detection event, allowing molecular identification of freely diffusing molecules in solution and detection of numerous targets simultaneously. Over the Phase I funding period, we will demonstrate feasibility by developing a test to simultaneously distinguish between 11 bacterial strains which are major pathogens responsible for nosocomial and community-acquired infections. Specific aims are: 1. Separate detection and quantification of eight genetic markers (species-specific for Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, E. faecium, and Shewanella oneidensis as positive control;resistance-specific for methicillin, vancomycin A, and vancomycin B) in purified DNA derived from MSSA, MRSA, VRSA, MSSE, MRSE, VSEfaecalis, vanA VREfaecalis, vanB VREfaecalis, VSEfaecium, vanA VREfaecium, vanB VREfaecium, and S. oneidensis by 3-color ALEX-based fluorescence spectroscopy. 2. Multiplexed (single-well) discrimination and quantification of bacteria spiked into human blood. 3. ALEX-based analysis of 350 archived clinical isolates from UCLA's Medical Center (including 25 positive for each of the 11 pathogen types, except for rare VRSA) and statistical analysis. PUBLIC HEALTH RELEVANCE: Nesher Technologies, Inc.'s proposed development of a highly multiplexed, ultrasensitive, quantitative, low- cost automated medical diagnostic test system, capable of quickly identifying specific healthcare-associated bacterial strains and drug resistance patterns among the multitude of possibilities from a single patient sample, radically pushes the limits of current technologies. The improved diagnostic information will enable physicians to better prescribe the appropriate antibiotics to patients and enable hospitals to implement infection control procedures to lower their infection rates, thereby saving human lives and reducing healthcare costs. This will allow limiting the use of broad-spectrum antibiotics, encourage the development of narrowly targeted therapeutics, and help curtailing the marked global trend towards increasing antibiotic drug resistance which is a major concern for treatment and management of infectious diseases, affecting the health of millions of people in the United States and around the world.